Herpesviruses permanently colonize their hosts by establishing latency. ?-herpesvirus infection is associated with cancer, especially in immunocompromised hosts, a fact related to establishment of latency in cells of the immune system. It is essential to define viral and host genes that control latency in order to understand, and ultimately manipulate chronic infection, a difficult task using pathogenesis studies in people. Thus we present this renewal application to study immune control of murine ?HV68 latency, a model that has already been used to identify principles and genetic effects that could not have been identified in other systems. We made progress on all initial aims leading to a novel testable hypothesis: that IFN3 regulates ?HV68 latency via effects on the critically important RTA promoter, thereby allowing the virus to utilize the host response to regulate its own gene expression program leading to stable chronic infection. Progress from the first grant period is reported in 13 manuscripts in PNAS, J. Virol., PLOS Pathogen, Virology, and Nature. We showed that it is possible to vaccinate against the establishment of ?- herpesvirus latency and defined the immunologic mechanisms responsible. Many studies led to the conclusion that the immune system controls latency via chronic expression of IFN3, which blocks ?HV68 reactivation from latency and regulates viral gene expression in vivo during chronic infection. A unique physiologic consequence of this continuous role for IFN3 is that latent herpesviruses can act as self-replicating innate immune adjuvants that define the set-point of innate immunity and the nature of the normal immune system. Preliminary data identifies the promoter for the essential latent-lytic switch protein RTA as one site for IFN3 effects on reactivation, supporting the concept that cell-extrinsic effects the host cytokine IFN3 determine the nature of latency and reactivation. This hypothesis is also supported by work from our group and others showing that IFN3 regulates latency for 1- and 2-herpesviruses. Regulation of herpesvirus latency in vivo by the cell-extrinsic factor IFN3 is likely important for human ?-herpesviruses based on IFN3 gene polymorphism associations with EBV associated post-transplant lymphoproliferative disease, and a case of overwhelming Kaposi's sarcoma in a child lacking the IFN3 receptor (IFN3R). What is/are the mechanism(s) responsible for these effects of IFN3? This is the question that will be answered by studies proposed in the following specific Aims. Aim 1. Test the hypothesis that IFN3 action on latently infected MF is necessary and sufficient to explain its effects on ?HV68 reactivation and pathogenesis. Aim 2. Test the hypothesis that multiple ?HV68 RTA promoters are IFN3 responsive. Aim 3. Test the hypothesis that IFN3 exerts cell-extrinsic control of ?HV68 reactivation via direct effects on viral RTA promoter(s). PUBLIC HEALTH RELEVANCE Many different herpesviruses colonize essentially all human beings for their entire lives by establishing a specialized infectious state called latency;this infection may be benign or may lead to cancer. In this study we will determine how the host's own immune system is taken advantage of by a gammaherpesvirus to allow the virus to remain in the host in a latent state. Understanding this mechanism for maintenance of latency may allow us to interfere with or control latency in an appropriate way, thus preventing the ill effects of chronic herpesvirus infection.